Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges

Abstract

Sickle cell disease (SCD) is a hereditary blood disorder caused by a specific mutation in the β-globin gene, leading to the production of hemoglobin S, which deforms red blood cells, causing occlusion in small blood vessels. This results in pain, anemia, organ damage, infections, and increased stroke risk. Treatment options, including disease-modifying therapies and curative hematopoietic stem cell transplants, have limited accessibility. Recently, autologous gene therapy has emerged as a promising curative option, particularly for SCD. Gene editing techniques such as CRISPR, base editing, and prime editing offer potential to correct this mutation. In this review, we discuss recent preclinical studies and clinical trials of gene and cell therapies, focusing on the progress of FDA-approved treatments like Lyfgenia and Casgevy. We also examine the many challenges, including accessibility, safety, and long-term efficacy, which continue to shape the future of SCD gene therapy.

Introduction

Millions worldwide are affected by sickle cell disease (SCD),¹ a hereditary blood disorder affecting individuals of African, Mediterranean, Middle Eastern, and South Asian descent.² The first molecular disease, as Linus Pauling called it, SCD is caused by a mutation in the β-globin gene (HBB) resulting in the production of hemoglobin S (HbS). This is an abnormal hemoglobin that affects the structure of red blood cells under deoxygenated conditions, resulting in the deformation of red blood cells, which in turn leads to occlusion in the small blood vessels.³ Downstream effects include severe pain episodes, anemia from destruction of these deformed red blood cells, end organ damage from vaso-occlusion, increased susceptibility to infections, and a risk of stroke.⁴ These complications lead to early morbidity and mortality in patients with SCD. In the United States, the median lifespan for individuals with SCD is 42 years for females and 38 years for males.²

Until recently, there were only a handful of treatment options for SCD available (Fig. 1), including disease-modifying therapies and curative options.⁵ Disease-modifying therapies include medications such as hydroxyurea, l-glutamine, Crizanlizumab, and Voxeletor. Around 25 years ago, the first cure for SCD was discovered when a pediatric patient with SCD underwent a hematopoietic stem cell transplant (HSCT) as part of leukemia treatment.⁶ Over time, this HSCT approach evolved into a viable strategy for addressing SCD, particularly among patients with Human Leukocyte Antigen-compatible siblings. However, HSCT is not an option for all patients with SCD given the lack of available donors and donors and the toxicities associated with conditioning regimens.⁷ HSCT is also often associated with complications such as graft-versus-host disease (GVHD) and the potential for graft failure or rejection.

FIG. 1.

Current treatment options, processes and timeline available to patients with SCD from beginning to end. SCD, sickle cell disease

For these reasons and due to the continual advances in technology, autologous gene therapy has come to the forefront of curative options for sickle cell patients. SCD results from a single point mutation in the HBB gene, which causes a substitution from glutamic acid (E) to valine (V) at the sixth amino acid (E6V), resulting in HbS instead of the wild-type Hb (HbA).⁸ As SCD is a monogenic disease, autologous gene therapy is a viable option to treat this condition. There are several different editing techniques being explored, some of which have been successfully delivered to patients, while others remain in development.^7,9–11 These techniques include the correction of the underlying mutation in the HBB gene, induction of fetal hemoglobin (HbF) to rescue patients from the sickling phenotype, and the addition of a functional anti-sickling β-globin gene.¹²

In this review, we shall focus on gene therapy for patients with SCD from preclinical studies to clinical trials and the recent Food and Drug Administration (FDA) approval (in December 2023) of two landmark therapies for SCD. We will also review access to care and hurdles associated with the delivery of these products to patients.

Gene Editing Techniques

The desired outcome in gene editing is to take a DNA sequence and convert it into a new desired sequence. The selection of the method used will depend on the desired edit.¹³ In SCD, several targets have been explored and found to be advantageous. The first is the HBB gene itself, while another commonly studied target is BCL11A, a repressor of γ-globin expression.¹⁴ These genes can be edited to correct the mutation using techniques such as Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9), base editing, and prime editing. In this review, we will focus on the techniques that have been applied clinically.

Prior to the emergence of CRISPR-Cas technology, zinc finger nucleases (ZFNs) were used to study SCD.¹⁵ ZFNs link the DNA-binding domain of zinc-finger proteins (ZFPs) with the nuclease domain of the FokI restriction enzyme.¹⁶ ZFPs consist of a tandem array of Cys2-His2 finger motifs, enabling ZFNs to bind to short DNA sequences. Each ZFP domain targets a specific DNA triplet, and ZFNs work as a pair of nucleases that recognize sequences on both the forward and reverse DNA strands surrounding the target site, binding to each side. Once bound, the FokI domain pair induces a double-stranded break (DSB) at the target site, cleaving the DNA.¹⁷

However, ZFNs have limitations in targeting specific DNA sequences, and the lack of specificity in some zinc-finger domains can result in off-target cleavage. Moreover, using ZFNs to target precise DNA sequences is a complex and labor-intensive process.^18,19 This has led to a focus on CRISPR and other more accessible techniques. Bacteria use RNA-guided endonucleases of CRISPR-Cas systems to recognize and cleave foreign nucleic acids. They retain a record of previously encountered pathogens by capturing nucleic acid sequences known as “spacer sequences” for future encounters.²⁰ Using different spacer sequences within a guide RNA, we can reprogram these systems to target DNA and RNA sequences as long as the matching target DNA protospacer sequence is located next to a suitable protospacer-adjacent motif (PAM).²¹ Cas9 effectors are RNA-guided endonucleases that generate DSBs in target DNA sequences.²² These breaks are then repaired by different DNA repair machinery that are endogenous to cells.

Another form of editing involves the addition of a functional gene to compensate for the loss of a functional β-globin gene. This includes the addition of functional genes that can be delivered to the genome by means of a viral vector.²³ There are several viral vectors that can be used such as lentivirus and adeno-associated virus (AAV). This technique, known as transduction, has wide applicability for cell types, high transfection rate, minimal immunogenicity, and minimal cytotoxicity.²⁴ However, there are packaging restrictions depending on the vector selected.²⁵

Building on the CRISPR platform, base editors have emerged as a very promising technology to target and modify single-base mutations. They do not require the formation of DSBs or donor DNA template as is required using CRISPR.¹³ As a result, fewer deleterious insertions and deletions are created. Base editing involves a nonfunctioning CRISPR-Cas nuclease combined with a single-stranded DNA deaminase. Base editors can efficiently mediate all four possible transition mutations (C→T, A→G, T→C, G→A), which represent approximately 30% of currently annotated human pathogenic variants.

Prime editing is another precision genome editing technique that is being studied in the context of SCD correction.^26–28 The prime editor protein is a combination of Cas9 and a highly engineered reverse transcriptase (RT), which works together with a prime editing guide RNA (pegRNA). The pegRNA targets the specific editing site and encodes the necessary edit. Prime editing enables precise insertions, deletions, and all 12 types of point mutations with minimal off-target effects, as the correction is directly transcribed from the pegRNA by RT.¹³

While gene editing techniques offer groundbreaking potential for treating SCD, their efficacy and safety have been rigorously tested through pivotal preclinical studies that have laid the foundation for clinical application.

Pivotal Preclinical Studies

Various therapeutic strategies are being investigated for treating SCD, including gene addition, gene editing, and gene regulation techniques.¹² The path to FDA approval for gene therapies targeting SCD has been marked by groundbreaking preclinical trials that established the safety and efficacy of these treatments. Here, we will focus on the pivotal preclinical studies related to autologous HSCT.

Lyfgenia is a gene therapy developed by Bluebird Bio that involves the use of a lentiviral vector to insert a functional copy of the β-globin gene into a patient’s hematopoietic stem cells.²⁹ The goal is to compensate for the underlying genetic defect by means of gene addition. Multiple preclinical studies have reported the approach using the βT87Q-globin gene to reduce the production of HbS in red blood cells. It was tested in differentiated human erythroid cells ex vivo and demonstrated a significant reduction in HbS levels and improved anti-sickling properties.³⁰ In 2001, Leboulch et al. supplied in vivo data in genetically modified mice that model the human condition.³¹ By employing a gene therapy approach that involves introducing a functional copy of the β-globin gene into mouse hematopoietic stem cells, researchers achieved significant restoration of normal hemoglobin production and a reduction in HbS levels. The treated mice showed improved red blood cell function and reduced disease symptoms, demonstrating that the gene therapy effectively addressed the genetic defect underlying SCD. These findings suggested that βT87Q-globin gene therapy could effectively mitigate SCD by decreasing HbS production and enhancing red blood cell function, paving the way for clinical applications.

Gene editing technologies, particularly CRISPR-Cas9, have also played a pivotal role in developing new therapies for SCD, with particular emphasis on editing the BCL11A repressor. This approach led to clinical trials (sponsored by Vertex Pharmaceuticals and CRISPR therapeutics) for Casgevy, the first CRISPR-Cas9 therapy available to patients with SCD. This gene editing technique focuses on disrupting the BCL11A repressor of HbF production, which is normally shut down in the months after birth.^23,33 Increasing HbF levels can alleviate SCD symptoms due to its anti-sickling properties, as evidenced by SCD patients who also carry mutations leading to HPFH, resulting in fewer or no symptoms.^11,34–36

Researchers have explored two pathways to upregulate HbF: a BCL11A knockout or targeting the BCL11A enhancer.³⁷ Direct knockout of the BCL11A gene leads to the complete loss of its function, ensuring a strong effect in terms of gene silencing. However, this may inadvertently disrupt nearby genes leading to off-target effects. Further, BCL11A is involved in other tissues, and its complete knockout could lead to adverse effects in nonerythroid cells, such as in the brain or other organs, where the gene appears to play a key role in development. Targeting the erythroid enhancer, which is specifically active in red blood cell precursors, should avoid off-target effects in nonerythroid cells^38,39 and offer a safer option.^40,41 However, targeting the enhancer might not completely shut down BCL11A, especially in cases where other compensatory mechanisms or enhancer elements exist that could mitigate the effect.

Ex vivo studies showed that genetic modification of human HSCs by BCL11A knockout led to increased HbF production and a reduction in sickling.^42–45 Researchers conducted studies using mouse models to investigate the effects of BCL11A knockout and showed increased expression of HbF.⁴⁶ Finally, to evaluate safety, other investigators evaluated the off-target effects of BCL11A editing using CRISPR.⁴⁷ Recent studies looking at dual editing of the BCL11A enhancer are showing promising results with higher levels of HbF induction.⁴⁰ This work has helped refine the CRISPR-Cas9 techniques to minimize risks and maximize the therapeutic potential, thus addressing concerns that could impact the transition to clinical trials.

While gene modulation techniques show promise, they do not address the underlying mutation of SCD. This can lead to ongoing hemolysis, as patients have been shown to produce 45% HbS, which has the potential for complications.^48,49 Correcting the sickle cell mutation directly within the patient’s own HSCs to produce normal hemoglobin has also been attempted, though it presents greater challenges than disruption methods.^50,51 Preclinical studies have shown promise for this technique, but the first trial testing this approach (sponsored by Graphite Bio) was halted due to an adverse event in the initial patient.⁵² This patient has since been followed by Kamau Therapeutics and found to have an initial increase in HbA followed by a downtrend around 7 months with a corresponding increase in HbF <78%. The mechanism of HbF upregulation is currently under investigation.⁵³

Newer techniques such as base and prime editing have also been studied in preclinical studies. Base editing cannot reverse the SCD point mutation to the wild-type sequence, but the use of an adenine base editor (ABE) produces an E6A genotype, known as Makassar beta-globin (HbG).⁵⁴ This is a rare naturally occurring variant that is considered nonpathogenic.^55,56 Studies have shown that ABEs can convert HbS to HbG in HEK293T cells with up to 55% efficiency.⁵⁷ Similarly, researchers using mouse models have achieved 80% editing from HbS to HbG using ex vivo delivery of ABEs into HSPCs derived from SCD patients.⁵⁸ Although Makassar β-globin holds promise as a potential therapeutic approach, further research in clinical trials is needed. Beam Therapeutics has chosen not to move this strategy into the clinic.⁵⁹ (Currently, there is no A-to-T base editor available for testing in SCD correction models.)

While base editing and prime editing are both emerging techniques, it is important to recognize when one might be more indicated than the other. Both techniques have their pros and cons.¹³ In some preclinical studies, base editors have been shown to have higher editing efficiencies in comparison to prime editors. Nonetheless, preclinical studies have demonstrated above 40% efficiency using prime editing, and in comparison to Cas9-HDR for HBB, some have shown 270-fold higher editing-to-indel ratio.²⁸

The success of these preclinical studies was pivotal in advancing both Lyfgenia and Casgevy to clinical trials. Both therapies received FDA approval in December 2023 for the treatment of SCD.^10,60 The positive outcomes in other preclinical models provided the necessary evidence of safety and efficacy required for initiating human trials that are now ongoing for the treatment of SCD.

Clinical Trials

In Tables 1 and 2, we list the gene therapy clinical trials for SCD based on two approaches: gene addition (Table 1) and gene editing (Table 2). Some trials on long-term effects are listed in Table 3. Here, we focus on trials that have resulted in FDA-approved treatments for SCD.

Table 1.

Gene addition clinical trials

Trial	Center	Title	Summary	Age eligibility (years)	Treatment	Accrual start date/status	Sponsor
NCT02151526	Single center, Interventional, Phase 1/2	A Study Evaluating the Safety and Efficacy of LentiGlobin BB305 Drug Product in β-Thalassemia Major (Also Referred to as Transfusion-dependent β-Thalassemia [TDT] and SCD HGB-205	Open label study evaluating the safety and efficacy of gene therapy of the β-hemoglobinopathies by transplantation of autologous CD34+ stem cells transduced ex vivo with a lentiviral β-A-T87Q Globin Vector (LentiGlobin BB305)	5–35	Lentiviral Vector	completed	Bluebird Bio
NCT02247843	Single center, UCLA, California Phase 1/2 study	Stem Cell Gene Therapy for SCD	Assess the safety and initial evidence for efficacy of an autologous transplant of lentiviral vector modified peripheral blood for adults with severe SCD	≥18	Lenti/G-βAS3-FB Lentiviral Vector	2014-12 Active, not recruiting	Donald B. Kohn (UCLA)
NCT02140554	Multicenter, Phase 1/2	A Study Evaluating the Safety and Efficacy of lovo-cel (bb1111) in Severe SCD	Study evaluating gene therapy by transplantation of autologous CD34+ stem cells transduced ex vivo with the LentiGlobin BB305 lentiviral vector in subjects with severe SCD	12–50	BB305 lentiviral vector Bb1111/ lovotibeglogene autotemcel/ Lovo-cel/ LentiGlobin BB305 Drug Product encoding the βA-T87Q-globin gene	2015-02-02, completed	Bluebird bio
NCT03282656	Multicenter, LA, Boston Phase 1	Gene Transfer for SCD	Determine the feasibility and safety of administering a lentiviral gene transfer vector encoding a small hairpin (sh) RNA targeting the γ-globin gene repressor, BCL11A, in patients with severe SCD.	2–40 (Stratum 1: ages ≥18–40 Stratum 2: ages ≥12-<18 Stratum 3: ages ≥3-<12)	lentiviral vector encoding a small hairpin (sh) RNA targeting BCL11A	2018-02-13, active not recruiting	David Williams (Boston Children’s Hospital)
NCT03964792	Single center, Paris, France A Phase 1/2	Safety and Efficacy of Gene Therapy of SCD by Transplantation of an Autologous CD34+ Enriched Cell Fraction That Contains CD34+ Cells Transduced ex Vivo with the GLOBE1 Lentiviral Vector Expressing the βAS3 Globin Gene in Patients with SCD (DREPAGLOBE) (DREPAGLOBE)	Open label study evaluating the safety and efficacy of gene therapy of SCD by transplantation of an autologous CD34+ enriched cell fraction that contains CD34+ cells transduced ex vivo with the GLOBE1 lentiviral vector expressing the βAS3 globin gene (GLOBE1 βAS3 modified autologous CD34+ Cells) in patients with SCD	12–20	Lentiviral vector, GLOBE1 ex vivo transduced, expressing an anti-sickling β-globin protein (AS3) containing three amino acid substitutions in the wild-type β-globin gene.	2019-11-12, active, not recruiting	Assistance Publique-Hôpitaux de Paris
NCT04293185	Multicenter, Phase 3	A Study Evaluating Gene Therapy with BB305 Lentiviral Vector in SCD	Study evaluating gene therapy by transplantation of autologous CD34+ stem cells transduced ex vivo with the BB305 lentiviral vector in subjects with SCD	2–50	autologous CD34+ cell-enriched population from patients with SCD that contains HSCs transduced with BB305 LVV (also knowns as bb1111) encoding the βA-T87Q-globin gene (lovotibeglogene autotemcel)	2020-02-14, recruiting	Bluebird bio
NCT05353647	Multicenter, Phase 2	A Gene Transfer Study Inducing Fetal Hemoglobin in SCD (GRASP, BMT CTN 2001) (GRASP)	Gene transfer study inducing fetal hemoglobin in SCD (GRASP, BMT CTN 2001)	13–40	lentiviral vector containing a shRNA targeting BCL11a	2022-07-12, recruiting	David Williams
NCT06399107	Single center, Beijing, China Phase 1/2	Investigation Into the Use of BAH243 Lentiviral Vector for Gene Therapy in Treating SCD (BAH243)	Lentiviral vector gene therapy in SCD using autologous CD34+ hematopoietic stem cells collected via apheresis and modified with a lentiviral vector	2–90	BAH243 lentiviral vector (LVV) carrying the human βA-T87Q-globin gene,	2024-08-01, recruiting	Essen Biotech

SCD, Sickle cell disease.

Table 2.

Gene editing clinical trials

Trial	Center	Title	Summary	Age eligibility (years)	Treatment	Accrual start date/status	Sponsor
NCT03745287	Multisite, Phase 1/2/3	A Safety and Efficacy Study Evaluating CTX001 in Subjects with Severe SCD	Study to evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9 Modified CD34+ human HSPCs (CTX001) in subjects with severe SCD	12–35	CTX001/ Exagamglogene autotemcel/ Exacel	2018-11-27, active, not recruiting	Vertex Pharmaceuticals
NCT03653247	Multicenter, Phase 1/2	A Study to Assess the Safety, Tolerability, and Efficacy of BIVV003 for Autologous Hematopoietic Stem Cell Transplantation in Patients with Severe SCD	Study to assess the safety, tolerability, and efficacy of BIVV003 cluster of differentiation 34+ HSPC (CD34+HSPC) transfected ex vivo with ZFN mRNAs targeting the BCL11A locus.	18–40	BIVV003	2019-03-06, active, not recruiting	Sangamo Therapeutics
NCT04443907	Multicenter Phase 1	Study of Safety and Efficacy of Genome-edited HSPCs in SCD	A first-in-patient Phase I/II clinical study to investigate the safety, tolerability and efficacy of genome-edited hematopoietic stem and progenitor cells in subjects with severe complications of SCD	2–40; Part A includes treatment of adults with OTQ923; Part B includes treatment of children (2–17) with OTQ923	OTQ923 or HIX763 (autologous CD34+ hHSPCs modified with CRISPR-Cas9 at the erythroid lineage-specific enhancer of BCL11A	2020-08-26, Terminated	Novartis Pharmaceuticals
NCT04853576	Multicenter, Phase 1/2	A Study Evaluating the Safety and Efficacy of EDIT-301 in Participants with Severe SCD (RUBY)	Study to evaluate the safety and efficacy of a single dose of autologous CRISPR gene-edited CD34+ human HSPCs (EDIT-301) in subjects with severe SCD	12–50	EDIT-301/ renizgamglogene autogedtemcel/reni-cel CRISPR-Cas12a/HBG1/2 edited in HSPCs	2021-05-04, active not recruiting	Editas Medicine
NCT04819841	Multisite (CA, Ohio, Missouri), Phase 1/2	Gene Correction in Autologous CD34+ Hematopoietic Stem Cells (HbS to HbA) to Treat Severe SCD (Restore)	Study of nula-cel in autologous CD34+ hematopoietic stem cells to convert HbS to HbA for Treating Severe SCD	>12–40	Nula-cel Drug Product is a human autologous CRISPR-Cas9 edited and sickle mutation-corrected HSPC product.	2021-11-15, recruiting	Kamau Therapeutics
NCT05329649	Multisite, Phase 3	Evaluation of Safety and Efficacy of CTX001 in Pediatric Participants with Severe SCD	Single-dose, open-label study in pediatric participants with severe SCD and hydroxyurea (HU) failure or intolerance. The study will evaluate the safety and efficacy of autologous CRISPR-Cas9 modified CD34+ HSPCs (CTX001)	2–11	Exa-cel/ Exagamglogene automcel/CTX001	Start accrual 2022-05-02, recruiting	Vertex Pharmaceuticals
NCT05477563	Multisite, Phase 3 b	Evaluation of Efficacy and Safety of a Single Dose of CTX001 in Participants with Transfusion-Dependent β-Thalassemia and Severe SCD	Study to evaluate efficacy and safety of a single dose of autologous CRISPR Cas9 Modified CD34+ human HSPCs (CTX001) in subjects with transfusion-dependent β-thalassemia or severe SCD	12–35	Exa-cel/CTX001 CRISPR-Cas9 edited at the BCL11A gene.	2022-08-02, recruiting	Vertex Pharmaceuticals
NCT05456880	Multicenter, Phase 1/2	BEACON: A Study Evaluating the Safety and Efficacy of BEAM-101 in Patients with Severe SCD	This is an open-label, single-arm, multicenter, study evaluating the safety and efficacy of the administration of autologous base edited CD34+ HSPCs (BEAM-101) in patients with severe SCD	18–35	Base editing BCL11A edited in HSPCs	2022-08-30, recruiting	Beam Therapeutics
NCT06287086	Single center	Clinical Study on the Safety and Efficacy of BRL-101 in the Treatment of SCD	Clinical study on the safety and efficacy of a single intravenous dose of CRISPR/Cas9-edited autologous CD34+ hematopoietic stem/progenitor cells (BRL-101) in the treatment of SCD	3–35	CRISPR-Cas9 edited at BCL11A gene (BRL-101)	2024-06-14, not yet recruiting	Bioray Laboratories
NCT06506461	Single center, Memphis, TN Phase 1	Gene Editing for SCD	Phase 1 study to assess the safety and efficacy of autologous infusion of clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated protein (Cas9)-edited CD34+ hematopoietic stem and progenitor cells (HSPCs) in patients with severe SCD.	>/=18 and </=24.9	(CRISPR)/ CRISPR associated protein (Cas9)-edited	2025-03-21	St. Jude’s Research Hospital
NCT06287099	Single center, (Nanfang Hospital, Southern Medical University)	Clinical Study of BRL-101 in the Treatment of SCD	Clinical study on the safety and efficacy of a single intravenous dose of CRISPR/Cas9-Edited autologous CD34+ hematopoietic stem/progenitor cells (BRL-101) in the treatment of SCD	3–35	BRL-101, Edited with CRISPR-Cas9 at the BCL11A gene.	Not yet recruiting, estimated 2024-04-20	Bioray Laboratories
NCT06300723	Single center	Clinical Study of BRL-101 in Severe SCD	Clinical study to evaluate the safety and efficacy of single dose intravenous infusion of CRISPR/Cas9-edited autologous CD34 + Hematopoietic Stem/Progenitor Cells (BRL-101) in the Treatment of Severe SCD	3–35	BRL -101, modified with CRISPR-Cas9 at the BCL11A gene.	2024-07-29, enrolling by invitation	Bioray Laboratories
NCT05951205	Multicenter, Phase 3	Evaluation of Efficacy and Safety of a Single Dose of Exa-cel in Participants with Severe SCD, βS/βC Genotype	Study to evaluate efficacy and safety of a single dose of exa-cel in subjects with severe SCD, βS/βC genotype	12–35	Exa-cel/CTX001 Edited with CRISPR-Cas9 at the BCL11A gene	Active, not yet recruiting	Vertex Pharmaceuticals

Source: clinicaltrials.gov.

Table 3.

Long-term follow-up trials

Trial	Center	Title	Summary	Age eligibility (years)	Treatment	Accrual start date/status	Sponsor
NCT05145062	Multisite, Observational	Long-term Follow-Up of SCD and Beta-thalassemia Subjects Previously Exposed to BIVV003 or ST-400	Follow-up study after ex vivo gene therapy with BIVV003 in participants with severe SCD or with ST-400 in Participants with Transfusion-dependent Beta-thalassemia (TDT) with Autologous HSCT	18–45	BIVV003	2024-04-16, recruiting	Sangamo Therapeutics
NCT06155500	Memphis, TN, Observational	Long-term Follow-up (LTFU) of Patients Treated with Genome-edited Autologous HSPCs	This study is monitoring patients treated with OTQ923 (Patients administered were OTQ923 while enrolled on the treatment protocol CADPT03A12101 (NCT04443907), Total of 15 years following infusion to monitor long-term safety and efficacy.	18+	OTQ923,	recruiting	Novartis
NCT04628585	Multicenter, Observational	Long-term Follow-up of Subjects with SCD Treated with Ex Vivo Gene Therapy	Follow-up study for subjects with SCD who have been treated with ex vivo gene therapy drug product in bluebird bio-sponsored clinical studies. Follow-up 15 years post-drug product infusion	2–53	Lentiviral vector	2020-10-21, enrolled by invitation	Bluebird bio
NCT04208529	Multisite, Phase 3	A Long-term Follow-up Study in Participants who Received CTX001	long-term safety and efficacy study of CTX001 in pediatric and adult participants who received CTX001 in parent studies 111 (NCT03655678) 141 (NCT05356195) or 161 (NCT05477563) (TDT studies) or Study 121 (NCT03745287) or 151 (NCT05329649), 161(NCT05477563),171 (NCT05951205) (severe SCD studies).	>2	CTX001	2021-01-20, recruiting	Vertex Pharmaceuticals

Following promising preclinical studies, Bluebird Bio conducted a single-center study (NCT02151526) evaluating the safety and efficacy of LentiGlobin, an autologous product transfected with the BB305 lentiviral vector Drug Product for patients with β-thalassemia or SCD. This trial included three patients (13, 16, and 21 years old) with severe SCD who safely received LentiGlobin. A follow-up after 5½ years continued to show sustained production of therapeutic HbAT87Q, along with a reduction of vaso-occlusive events and hemolysis.⁴⁹ These patients are now under long-term observation in a separate study (NCT04628585).

Based on these promising results, a multicenter study (NCT02140554) phase I/II, open label, nonrandomized study for patients with severe SCD was conducted in three cohorts. Cohort A included seven patients who received LentiGlobin manufactured from bone marrow-harvested CD34+ cells, which was durable but had a lower vector copy number and suboptimal expression of HbA. Significant changes were made for patients in group B, which included transfusions prior to collection to reduce stress erythropoiesis, a higher busulfan dose AUC (>5,000 µmol × min), and a refined manufacturing process to improve transduction efficacy. Group B consisted of two subgroups: Group B1 (n = 1) received total two products, one from previous manufacturing and one from refined process, and Group B2 (n = 1), who received products only from the refined process. These changes improved the quality and quantity of the collected cells as demonstrated by increased vector copy numbers and HbAT87Q production, higher hemoglobin levels, low HbS percentage, and decreased hemolysis.⁴⁹

The trial was briefly put on hold in 2021 following a report of two cases from Cohort A with acute myeloid leukemia (AML). Both patients received the product from bone marrow harvests and underwent the early manufacturing process. The first patient was diagnosed with myelodysplastic syndrome, which progressed to AML and death. Investigators determined it was unlikely from the drug product following absence of vector in blast population and attributed secondary to myeloablative chemotherapy. In the second case, vector was present in blast cells in the VAMP4 gene, which is not known to play a role in development of AML and considered unlikely to be a direct result of LentiGlobin.

The trial resumed with 35 patients in cohort C, which received LentiGlobin following further process optimization. This showed sustained and durable engraftment in all 35 patients with sustained pan-cellular expression of HbA^T87Q contributing to at least 40% of total hemoglobin and reduced to normalization of hemolysis markers. Two patients with two-gene deletion α-thalassemia trait treated showed persistent anemia with one adult requiring RBC transfusions. The hematological abnormalities were restricted to the erythroid lineage.⁴⁹ Among patients evaluated for transplant population with vaso-occlusive events (n = 25), all had resolution of severe vaso-occlusive events.

Vertex Pharmaceuticals and CRISPR Therapeutics conducted a multisite trial, CLIMB-SCD-121 using ex vivo CRISPR–Cas9 technology of gene editing autologous CD34+ HSPCs at the erythroid-specific enhancer region of BCL11A, leading to FDA approval of Casgevy. A total of 44 patients were enrolled in the CTX001 trial; most adverse events were noted secondary to chemotherapy, none from the infusion itself. The median of neutrophil engraftment was 27 days and 35 days for platelets. One death was reported due to SARS-CoV2 infection. In interim analysis, 30 patients met the primary efficacy endpoint and 29 (97%) were free of VOC for about 12 months. In the full analysis, of 43 patients, 37 (86%) were free from severe VOC for up to 45.5 months. There was a sustained increase in hemoglobin levels (12.5+/−1.8 g/dL) and fetal hemoglobin (43.9+/−8.6%) at month 6, and normal or near-normal levels at follow-up and with a mean HbF level of over 40% at follow-up. The mean percentage of F cells (the percentage of red cells expressing HbF) was greater than 90% at follow-up, with improvements of hemolysis markers. About 15 patients had a single α-globin gene deletion, two patients with two α-globin gene deletion; all of them showed similar increased total Hb and HbF levels in comparison with patients who did not have the α-globin gene deletion. The patients in this study will be followed in CLIMB-131 (NCT04208529) for long-term effects.

Although only Casgevy and Lyfgenia have received FDA approval so far, several other clinical trials are ongoing with promising data. Esrick et al.at Boston Children’s Hospital are conducting an ongoing study (NCT03282656) demonstrating a favorable risk-benefit profile in six patients with severe SCD, with a median follow-up of 18 months. Their gene editing approach uses short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR) via integration with a lentiviral vector. This successfully overcame BCL11A knockdown in other lineages by reducing erythroid-specific lineage cells by 90%. The median time for engraftment was 22 days for neutrophils and 33 days for platelets. Participants experienced substantial and stable increases in HbF levels (20–41% of total hemoglobin), with HbF broadly distributed across red blood cells (59–93.6% of cells contained HbF) and sustained throughout the study. None of the patients experienced VOC, acute chest syndrome, or stroke after treatment. Adverse events were primarily related to the chemotherapy conditioning regimen. While no insertional oncogenesis was noted, lentiviral therapies historically carry theoretical risks, as the BCH-BB694 vector requires genomic integration. Three patients who previously required regular blood transfusions for secondary stroke prevention were able to stop or reduce their transfusion regimen.⁶¹

Renizgamglogene autogedtemcel (reni-cel) is an investigational gene-edited product that uses the Cas12a nuclease to edit the promotor of the γ-globin genes, HBG1/HBG2 (−118 to −113 CCAAT box). This approach mimics mutations in patients with Hereditary Persistence of Fetal Hemoglobin and reactivates γ-globin expression. Preclinical studies have shown ≥80% editing efficiency in CD34+ cells with high specificity and no off-target editing.⁶² The RUBY trial (NCT04853576; sponsored by Editas Medicine) demonstrated early, durable correction of anemia with sustained HbF induction (≥40% total Hb with pancellular distribution) and normalization of hemolytic markers by month 6 in 28 patients with SCD, with a median follow-up of 9.5 months. Median engraftment times were 23 days for neutrophils and 25 days for platelets. By month 6, the mean percentage of F cells increased by 99.3%, with levels maintained above 97% throughout follow-up. Of 28 treated patients, 27 remained VOE-free post-infusion as of October 2024. The study also showed early and sustained meaningful improvements in pain, physical, and social patient-reported outcomes.⁶³

To understand the benefits of HbF induction via BCL11A inhibition compared with hydroxyurea, De Souza et al. used single-cell assays to show that BCH-BB694-treated patients had higher HbF percentages (27.9% vs. 27.0%) and fewer sickle-prone RBCs, defined as RBCs with HbS >70% (42% vs. 61%), compared with high responders to hydroxyurea.^64,65

LentiGlobin’s gene addition approach offers a direct replacement of defective hemoglobin, contrasting with the HbF-focused strategies of exa-cel, reni-cel, and BCH-BB694. While effective in reducing SCD complications, its reliance on lentiviral integration introduces safety considerations absent in CRISPR-based therapies. Exa-cel and reni-cel provide durable, precise DNA edits without viral vectors, positioning them as potentially safer long-term options despite similar efficacy profiles. CRISPR-based therapies induce permanent DNA edits, offering a likely advantage in long-term durability, while the long-term durability of shRNA-mediated BCL11A suppression for elevating HbF remains unknown.

Discussion

It has been 18 months since the landmark approval of two gene therapy products—Casgevy and Lyfgenia—for SCD. We are learning important principles and lessons that should guide us forward.

While we anticipate that it will get easier, it remains expensive, difficult, and time-consuming to progress from initial patient consult to curative therapy. Authorization of these products can take many weeks and frequently requires a single case agreement with the payor. Research, regulatory, and manufacturing costs have led to biotech companies setting prices of more than $2 million per product. Before scheduling patients for stem cell collection, authorization must be in place. On a positive note, as of December 2024, both manufacturers (Vertex and Bluebird) had entered into agreements with the Centers for Medicare & Medicaid Services to participate in the Cell and Gene Therapy Access Model. States participating in outcomes-based models for their patients with Medicaid will likely be able to lower the overall cost of delivery of gene therapy.⁶⁶

In addition, patients with SCD frequently require more than one stem cell collection. One reason is that filgrastim mobilization cannot be used given the risk of VOC and mobilization with plerixafor alone is not always as effective.⁶⁷ Second, even with an adequate stem cell dose, there is often insufficient production of the genetically manufactured product, requiring additional collections. For example, there can be significant loss of stem cells during the electroporation process (used in Casgevy).

Once we send a product for manufacturing with an adequate cell dose, there are several possible outcomes: (1) the center receives genetically modified product that meets cell dose requiring no additional collections; (2) the center receives genetically modified product that does not meet the required cell dose, necessitating additional collection; and (3) genetic manipulation was not successful or the product is deemed out of specification, requiring full recollection. It can take 3–5 months to progress from the last collection to infusion (Fig. 1). During that time, the patient may continue experiencing symptoms such as VOC and acute chest syndrome. Given this complex patient journey⁶⁸ requiring insurance approval, multiple stem cell collections, and centers navigating coordination with apheresis, clinical teams, interventional radiology, flow lab, cell therapy lab, and the sponsor, it is no surprise why fewer treatments have been performed to date than was anticipated and hoped for by the companies and the SCD community.

The overall treatment, including transfusions, mobilization, sickle cell care, pain management, mental health support, and delivery of high-dose chemotherapy must occur at a comprehensive medical center capable of handling and coordinating all aspects of patient care. Our patients will not be served well by an excellent sickle cell program without an experienced transplant program and vice versa. The patients coming to gene therapy are some of the most complex sickle cell patients and require very specialized multidisciplinary care. In terms of their myeloablative chemotherapy, using high doses of busulfan, the risks not only of mucositis and nausea/vomiting are high; in addition, the risk of sinusoidal obstruction syndrome is significant enough requiring comfort in the initiation of defibrotide and the use of diuretic drips and/or dialysis as needed.⁶⁹

Although we are still in the infancy of this field, if results continue to be successful, uptake of gene therapy will increase as patients, payors, and centers become more comfortable offering this therapy. For now, it is offered as an alternative to haploidentical transplant in eligible patients over 12 years of age, but one may imagine that given no risk of GVHD and lower risks of infection that gene therapy may eventually continue gaining ground.

Alternative donor transplants have become widely accessible (90% donor availability) with impressive outcomes (88% event-free survival, 95% overall survival at two years).⁷⁰ They remain the standard of care globally, especially where gene therapy is cost-prohibitive. Transplants work effectively even for patients with organ damage through reduced-intensity protocols.

Gene therapy, while eliminating donor needs and GVHD risks, costs 5–6 times more than transplantation ($2–3 million vs. $467,747) and often excludes patients with organ damage due to intensive chemotherapy requirements. Despite current limitations, gene therapy could revolutionize global treatment if made more affordable and accessible. Both approaches offer transformative potential, with treatment selection depending on individual patient factors including donor availability, existing organ damage, and financial considerations.

Over the next 5–10 years, we envision certain aspects of the therapy and its delivery to change, allowing for better access and more patients treated. We expect studies to show the treatment is beneficial in patients younger than 12 as well as in patients with stroke or cerebrovascular events. This will open the door to many new patients. As we have more patients willing to go through the treatment, the companies will increase the manufacturing slots. Both pharmaceutical companies and academia need to focus on certain issues to broaden applicability. Biopharma companies must continue working on their manufacturing process with a goal of reducing cell loss and reducing out-of-specification products. This will hopefully result in many more patients having successful products after a single collection. Academic centers should focus on trials with less-toxic chemotherapy (such as treosulfan,⁷¹ which was recently approved by the FDA), which will lead to milder short term side effects and possibly less infertility. Additionally, if studies with the stem cell antibody CD117⁷² show success, this would revolutionize the field as patients are vocal about their concerns of side effects with myeloablative chemotherapy, particularly infertility.

Over the next decade, however, we envision the field shifting to in vivo gene therapy. There are promising preclinical studies looking at the delivery of genetic material using AAV vectors or liquid nanoparticles.⁷³ The approach may be similar to one used by neurologists with Elevidys used to treat Duchenne muscular dystrophy.⁷⁴ Dispensing with chemotherapy would be welcome but there are open questions: will there be unintended delivery to cells other than hematopoietic stem cells? How long does the “cure” last with this approach and how frequently would re-dosing be needed?

Conclusions

The advent of gene therapy for SCD marks a transformative shift in the landscape of treatment options for this debilitating genetic disorder. Despite the availability of disease-modifying therapies like hydroxyurea, the only true curative option until recently was a HSCT, which remains inaccessible for many patients due to the challenges of finding suitable donors and the risks associated with the procedure. Autologous gene therapy has risen to the forefront as a promising cure, leveraging the ability to correct or modify the patient’s own genetic material.

Gene therapy approaches focus on three main strategies: correcting the underlying mutation in the β-globin gene; inducing HbF production; and adding functional globin genes. Technologies like CRISPR, base editing, and prime editing offer a means to precisely modify genes involved in disease pathology. Each technique has its own advantages and challenges, but collectively, they represent groundbreaking progress in the search for a cure.

As summarized earlier, a number of preclinical studies have demonstrated the potential of these therapies in both animal models and ex vivo human cell cultures. FDA-approved treatments like Lyfgenia and Casgevy are a testament to the potential of gene therapy in treating SCD. These therapies have demonstrated remarkable results, including the resolution of VOC, normalization of hemoglobin levels, and significant reductions in disease-related complications. The success of these treatments in clinical trials highlights the promise of gene therapy not only as a curative option but also as a long-term solution that offers patients the possibility of a life free from the debilitating symptoms of SCD. However, the path to these historic FDA approvals was not without challenges, including concerns about the long-term safety of gene therapies, such as potential off-target effects and the risks associated with the conditioning regimens used prior to therapy.

Despite the promise shown by these therapies in the clinic, several hurdles remain. One of the primary challenges is accessibility, as these treatments are complex, costly, and require specialized infrastructure. Additionally, the process of stem cell collection, genetic modification, and reinfusion can take many months, during which patients may continue to suffer from the symptoms of the disease. Furthermore, the need for long-term follow-up to assess the durability of treatment outcomes and manage any potential late-onset complications is critical. As the field evolves, efforts to improve manufacturing efficiency, reduce the cost of treatment, and expand access to care will be crucial in ensuring that these therapies reach a broader patient population.

Looking ahead, the future of gene therapy for SCD appears promising. Continued research is necessary to refine gene editing techniques, improve manufacturing processes, and enhance the safety and accessibility of these treatments. Moreover, as more clinical trials expand the patient populations eligible for gene therapy, including younger patients and those with complications like stroke, the scope of gene therapy’s impact will only grow. With ongoing advancements and a focus on improving patient outcomes, gene therapy could become the standard of care for SCD, offering hope to millions affected by this devastating disease.

Footnotes

Acknowledgment

The authors thank Talia Jacobsohn for assistance with editing .

FIG. 2.

Barriers to care in gene therapy.

Authors’ Contributions

All authors wrote sections of the article, and they all edited and approved of the final version. D.J. and H.B. created the figures; M.M. created the tables. All authors edited and approved the final version.

Author Disclosure Statement

D.J. served on an advisory board meeting for Vertex Pharmaceuticals in 2024.

Funding Information

No funding was received for this article.

References

Elendu

, Amaechi

, Alakwe-Ojimba

, et al. Understanding Sickle cell disease: Causes, symptoms, and treatment options. Medicine (Baltimore), 2023; 102(38):e35237; doi: 10.1097/MD.0000000000035237

Rees

, Williams

, Gladwin

. Sickle-cell disease. Lancet, 2010; 376(9757):2018–2031; doi: 10.1016/S0140-6736(10)61029-X

Bunn

. Pathogenesis and treatment of sickle cell disease. N Engl J Med, 1997; 337(11):762–769; doi: 10.1056/NEJM199709113371107

Platt

, Brambilla

, Rosse

, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med, 1994; 330(23):1639–1644; doi: 10.1056/NEJM199406093302303

Abdel-Hadi

, Ventura Carmenate

, Castillo-Aleman

, et al. Treatment of sickle cell disease - options and perspective. Am J Blood Res, 2023; 13(2):61–70.

Fitzhugh

, Abraham

, Tisdale

, et al. Hematopoietic stem cell transplantation for patients with sickle cell disease: Progress and future directions. Hematol Oncol Clin North Am, 2014; 28(6):1171–1185; doi: 10.1016/j.hoc.2014.08.014

Leonard

, Tisdale

, Bonner

. Gene therapy for hemoglobinopathies: Beta-thalassemia, sickle cell disease. Hematol Oncol Clin North Am, 2022; 36(4):769–795; doi: 10.1016/j.hoc.2022.03.008

Pinto

, Balocco

, Quintino

, et al. Sickle cell disease: A review for the internist. Intern Emerg Med, 2019; 14(7):1051–1064; doi: 10.1007/s11739-019-02160-x

Williams

, Esrick

. Investigational curative gene therapy approaches to sickle cell disease. Blood Adv, 2021; 5(23):5452; doi: 10.1182/bloodadvances.2021005567

10.

Harris

. Sickle cell disease approvals include first CRISPR gene editing therapy. JAMA, 2024; 331(4):280; doi: 10.1001/jama.2023.26113

11.

Demirci

, Uchida

, Tisdale

. Gene therapy for sickle cell disease: An update. Cytotherapy, 2018; 20(7):899–910; doi: 10.1016/j.jcyt.2018.04.003

12.

Hoban

, Orkin

, Bauer

. Genetic treatment of a molecular disorder: Gene therapy approaches to sickle cell disease. Blood, 2016; 127(7):839–848; doi: 10.1182/blood-2015-09-618587

13.

Anzalone

, Koblan

, Liu

. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol, 2020; 38(7):824–844; doi: 10.1038/s41587-020-0561-9

14.

Yin

, Xie

, Ye

, et al. BCL11A: A potential diagnostic biomarker and therapeutic target in human diseases. Biosci Rep, 2019; 39(11); doi: 10.1042/BSR20190604

15.

Klug

. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev Biochem, 2010; 79:213–231; doi: 10.1146/annurev-biochem-010909-095056

16.

Sun

, Zhao

. A single-chain TALEN architecture for genome engineering. Mol Biosyst, 2014; 10(3):446–453; doi: 10.1039/c3mb70412b

17.

Urnov

, Rebar

, Holmes

, et al. Genome editing with engineered zinc finger nucleases. Nat Rev Genet, 2010; 11(9):636–646; doi: 10.1038/nrg2842

18.

Ramirez

, Foley

, Wright

, et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods, 2008; 5(5):374–375; doi: 10.1038/nmeth0508-374

19.

Pattanayak

, Ramirez

, Joung

, et al. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods, 2011; 8(9):765–770; doi: 10.1038/nmeth.1670

20.

Mojica

, Diez-Villasenor

, Garcia-Martinez

, et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol, 2005; 60(2):174–182; doi: 10.1007/s00239-004-0046-3

21.

Mojica

, Diez-Villasenor

, Soria

, et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol, 2000; 36(1):244–246; doi: 10.1046/j.1365-2958.2000.01838.x

22.

Pourcel

, Salvignol

, Vergnaud

. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading), 2005; 151(Pt 3):653–663; doi: 10.1099/mic.0.27437-0

23.

Demirci

, Essawi

, Germino-Watnick

, et al. Advances in CRISPR Delivery Methods: Perspectives and Challenges. CRISPR J, 2022; 5(5):660–676; doi: 10.1089/crispr.2022.0051

24.

Taha

, Lee

, Hotta

. Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges. J Control Release, 2022; 342:345–361; doi: 10.1016/j.jconrel.2022.01.013

25.

Bulcha

, Wang

, Ma

, et al. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther, 2021; 6(1):53; doi: 10.1038/s41392-021-00487-6

26.

Matsoukas

. Prime editing: Genome editing for rare genetic diseases without double-strand breaks or donor DNA. Front Genet, 2020; 11:528; doi: 10.3389/fgene.2020.00528

27.

Anzalone

, Randolph

, Davis

, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019; 576(7785):149–157; doi: 10.1038/s41586-019-1711-4

28.

Everette

, Newby

, Levine

, et al. Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice. Nat Biomed Eng, 2023; 7(5):616–628; doi: 10.1038/s41551-023-01026-0

29.

Kanter

, Walters

, Krishnamurti

, et al. Biologic and clinical efficacy of LentiGlobin for sickle cell disease. N Engl J Med, 2022; 386(7):617–628; doi: 10.1056/NEJMoa2117175

30.

Demirci

, Gudmundsdottir

, Li

, et al. betaT87Q-Globin gene therapy reduces sickle hemoglobin production, allowing for ex vivo anti-sickling activity in human erythroid cells. Mol Ther Methods Clin Dev, 2020; 17:912–921; doi: 10.1016/j.omtm.2020.04.013

31.

Pawliuk

, Westerman

, Fabry

, et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science, 2001; 294(5550):2368–2371; doi: 10.1126/science.1065806

32.

Liu

, Hargreaves

, Zhu

, et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell, 2018; 173(2):430–442 e17; doi: 10.1016/j.cell.2018.03.016

33.

Weber

, Frati

, Felix

, et al. Editing a gamma-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Sci Adv, 2020; 6(7); doi: 10.1126/sciadv.aay9392

34.

Kunz

, Kulozik

. Gene therapy of the hemoglobinopathies. Hemasphere, 2020; 4(5):e479; doi: 10.1097/HS9.0000000000000479

35.

, Wang

, Tan

, et al. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and beta-thalassemia. Proc Natl Acad Sci U S A, 2016; 113(38):10661–10665; doi: 10.1073/pnas.1612075113

36.

, Yang

, Peng

, et al. CRISPR/Cas9-based gene-editing technology for sickle cell disease. Gene, 2023; 874:147480; doi: 10.1016/j.gene.2023.147480

37.

Canver

, Smith

, Sher

, et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature, 2015; 527(7577):192–197; doi: 10.1038/nature15521

38.

Bauer

, Kamran

, Lessard

, et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science, 2013; 342(6155):253–257; doi: 10.1126/science.1242088

39.

Smith

, Luc

, Croney

, et al. Strict in vivo specificity of the Bcl11a erythroid enhancer. Blood, 2016; 128(19):2338–2342; doi: 10.1182/blood-2016-08-736249

40.

Demirci

, Zeng

, Palchaudhuri

, et al. BCL11A +58/+55 enhancer-editing facilitates HSPC engraftment and HbF induction in rhesus macaques conditioned with a CD45 antibody-drug conjugate. Cell Stem Cell, 2025; 32(2):209–226.e8; doi: 10.1016/j.stem.2024.10.014

41.

Vierstra

, Reik

, Chang

, et al. Functional footprinting of regulatory DNA. Nat Methods, 2015; 12(10):927–930; doi: 10.1038/nmeth.3554

42.

Dever

, Bak

, Reinisch

, et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature, 2016; 539(7629):384–389; doi: 10.1038/nature20134

43.

, Zeng

, Roscoe

, et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat Med, 2019; 25(5):776–783; doi: 10.1038/s41591-019-0401-y

44.

Traxler

, Yao

, Wang

, et al. A genome-editing strategy to treat beta-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med, 2016; 22(9):987–990; doi: 10.1038/nm.4170

45.

Lamsfus-Calle

, Daniel-Moreno

, Antony

, et al. Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34(+) HSPCs by CRISPR/-Cas9 for the induction of fetal hemoglobin. Sci Rep, 2020; 10(1):10133; doi: 10.1038/s41598-020-66309-x

46.

, Bauer

, Kerenyi

, et al. Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A. Proc Natl Acad Sci U S A, 2013; 110(16):6518–6523; doi: 10.1073/pnas.1303976110

47.

Bjurstrom

, Mojadidi

, Phillips

, et al. Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases. Mol Ther Nucleic Acids, 2016; 5(8):e351; doi: 10.1038/mtna.2016.52

48.

Frangoul

, Locatelli

, Sharma

, et al.; CLIMB SCD-121 Study Group. Exagamglogene Autotemcel for Severe Sickle Cell Disease. N Engl J Med, 2024; 390(18):1649–1662; doi: 10.1056/NEJMoa2309676

49.

Kanter

, Thompson

, Pierciey

Jr , et al. Lovo-cel gene therapy for sickle cell disease: Treatment process evolution and outcomes in the initial groups of the HGB-206 study. Am J Hematol, 2023; 98(1):11–22; doi: 10.1002/ajh.26741

50.

Frangoul

, Altshuler

, Cappellini

, et al. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia. N Engl J Med, 2021; 384(3):252–260; doi: 10.1056/NEJMoa2031054

51.

Lattanzi

, Camarena

, Lahiri

, et al. Development of beta-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease. Sci Transl Med, 2021; 13(598); doi: 10.1126/scitranslmed.abf2444

52.

Yao

. Graphite Bio announces voluntary pause of phase 1/2 CEDAR study of nulabeglogene autogedtemcel (nula-cel) for sickle cell disease. 2023.

53.

Shyr

, Miller

, Schroeder

, et al. One year follow-up on the first patient treated with Nula-Cel: An autologous CRISPR/Cas9 gene corrected CD34+ Cell product to treat sickle cell disease. Blood, 2023; 142(Supplement 1):5000–5000; doi: 10.1182/blood-2023-188963

54.

Chu

, Packer

, Rees

, et al. Rationally designed base editors for precise editing of the sickle cell disease mutation. CRISPR J, 2021; 4(2):169–177; doi: 10.1089/crispr.2020.0144

55.

Mohamad

, Hamzah

, Selvaratnam

, et al. Human hemoglobin G-Makassar variant masquerading as sickle cell anemia. Hematol Rep, 2018; 10(3):7210; doi: 10.4081/hr.2018.7210

56.

Viprakasit

, Wiriyasateinkul

, Sattayasevana

, et al. Hb G-Makassar [beta6(A3)Glu–>Ala; codon 6 (GAG–>GCG)]: molecular characterization, clinical, and hematological effects. Hemoglobin, 2002; 26(3):245–253; doi: 10.1081/hem-120015028

57.

Miller

, Wang

, Randolph

, et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol, 2020; 38(4):471–481; doi: 10.1038/s41587-020-0412-8

58.

Newby

, Yen

, Woodard

, et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature, 2021; 595(7866):295–302; doi: 10.1038/s41586-021-03609-w

59.

Therapeutics B. Beam therapeutics announces first development candidates for sickle cell disease and reports second quarter 2020 results. Globalnewswire, 2020.

60.

Davies

, Philippidis

, Barrangou

. Five years of progress in CRISPR clinical trials (2019–2024). Crispr J, 2024; 7(5):227–230; doi: 10.1089/crispr.2024.0081

61.

Esrick

, Lehmann

, Biffi

, et al. Post-transcriptional genetic silencing of BCL11A to treat sickle cell disease. N Engl J Med, 2021; 384(3):205–215; doi: 10.1056/NEJMoa2029392

62.

Hanna

, McKinney

, Pineiro

, et al. AsCas12a gene editing of HBG1/2 promoters with EDIT-301 results in rapid and sustained normalization of hemoglobin and increased fetal hemoglobin in patients with severe sickle cell disease and transfusion-dependent beta-thalassemia. Blood, 2023; 142(Supplement 1):4996–4996; doi: 10.1182/blood-2023-187397

63.

Hanna

, McKinney

, Pineiro

, et al. Reni-Cel, an investigational AsCas12a gene-edited cell medicine, led to sustained hemoglobin normalization and increased fetal hemoglobin in patients with severe sickle cell disease treated in the RUBY trial. blood, 2024; 144(Supplement 1):4955–4955; doi: 10.1182/blood-2024-210343

64.

De Souza

, Hebert

, Esrick

, et al. Genetic reversal of the globin switch concurrently modulates both fetal and sickle hemoglobin and reduces red cell sickling. Nat Commun, 2023; 14(1):5850; doi: 10.1038/s41467-023-40923-5

65.

Marisa Wexler

. Gene therapy lowers number of red blood cells that take sickle shape-Proportion of cells that make abnormal HbS decline with BCH-BB694 treatment. 2023.

66.

Services CfMM. Biden-Harris Administration Takes Next Steps to Increase Access to Sickle Cell Disease Treatments. cms.gov; 2024.

67.

Leonard

, Sharma

, Uchida

, et al. Disease severity impacts plerixafor-mobilized stem cell collection in patients with sickle cell disease. Blood Adv, 2021; 5(9):2403–2411; doi: 10.1182/bloodadvances.2021004232

68.

Gray

, Thomas

, Davies

. Warrior spirit: An interview with victoria gray, sickle cell pioneer. CRISPR J, 2024; 7(1):5–11; doi: 10.1089/crispr.2024.29171.vgr

69.

Dignan

, Wynn

, Hadzic

, et al.; British Society for Blood and Marrow Transplantation. BCSH/BSBMT guideline: Diagnosis and management of veno-occlusive disease (sinusoidal obstruction syndrome) following haematopoietic stem cell transplantation. Br J Haematol, 2013; 163(4):444–457; doi: 10.1111/bjh.12558

70.

Kassim

, Walters

, Eapen

, et al. Haploidentical bone marrow transplantation for sickle cell disease. NEJM Evid, 2025; 4(3):EVIDoa2400192; doi: 10.1056/EVIDoa2400192

71.

Aygüneş

, Karagun

, Ay Tuncel

, et al. Busulfan-based and treosulfan-based myeloablative conditioning for allogeneic transplantation in children with thalassemia major: A single-center experience from Southern Turkey. Exp Clin Transplant, 2023; 21(11):883–892; doi: 10.6002/ect.2023.0143

72.

Uchida

, Stasula

, Demirci

, et al. Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model. Nat Commun, 2023; 14(1):6291; doi: 10.1038/s41467-023-41153-5

73.

Volodina

, Smirnikhina

. The future of gene therapy: A review of in vivo and ex vivo delivery methods for genome editing-based therapies. Mol Biotechnol, 2025; 67(2):425–437; doi: 10.1007/s12033-024-01070-4

74.

Hoy

. Delandistrogene moxeparvovec: First approval. Drugs, 2023; 83(14):1323–1329; doi: 10.1007/s40265-023-01929-x